Borrelia burgdorferi, the infectious agent of Lyme disease, is transmitted to mammals through the bite of infected Ixodes ticks. Our broad objective is to use a genetic approach to elucidate the molecular mechanisms of adaptation and variation in B. burgdorferi and their roles in the infectious cycle. We have developed a method of gene inactivation by allelic exchange, using as a selectable marker a mutated form of an endogenous borrelial gene, gyrB, which confers resistance to the antibiotic coumermycin. While initially invaluable as a marker to follow transformation, the utility of the coumermycin- resistant gyrB gene for other purposes in B. burgdorferi is severely limited by the high frequency at which it recombines with the endogenous gyrB gene. We have recently developed another selectable marker that does not have this shortcoming. We have integrated a large segment of foreign DNA including a kanamycin-resistance gene (Kan) into B. burgdorferi. The resulting integrant remained sensitive to kanamycin and we subsequently demonstrated that this foreign kan gene is poorly expressed from its native promoter in B. burgdorferi. We have since reconstructed the kan gene by fusing it to the promoter from either of two constitutively expressed borrelial genes and demonstrated that B. burgdorferi transformants carrying this gene are resistant to kanamycin. In the short period since it was developed, this marker has greatly facilitated our efforts to inactivate genes, introduce foreign DNA and improve methods of transformation in B. burgdorferi and should be increasingly valuable in future studies. Our desire to analyze the phenotype of mutant spirochetes in the infectious cycle is hampered by our current inability to transform infectious spirochetes. All successful gene inactivations to date have been in an attenuated, non- infectious variant of the type strain B31, which is easily transformed by electroporation. Numerous attempts to transform an infectious clone of strain B31 by this method have been unsuccessful. We have taken two different approaches to circumvent this problem. The first is to use alternative methods for transformation of the type strain B31. The second is to use a different strain, N40, which is both infectious and easily resuspended in electroporation solution. We have not yet succeeded in introducing mutations into an infectious clone, but we have just begun to use the kan gene as a selectable marker. We also still lack a shuttle vector for genetic studies in B. burgdorferi. In addition to their utility for this purpose, identifying sequences required for plasmid replication in B. burgdorferi will provide information about this unique component of the borrelial genome. We have tested DNA fragments encompassing the chromosomal origin of replication and conserved plasmid genes for their ability to confer autonomous replication. We have also cloned several large segments of borrelial plasmids, as well as an entire small borrelial plasmid, into an E. coli vector and will test their utility as a shuttle vector. Along with many other uses, the kanamycin-resistance marker will greatly simplify and facilitate development of a shuttle vector. - Borrelia burgdorferi, spirochete, Lyme disease, molecular genetics, tick-borne